This is a proposal for a comprehensive study of the molecular basis of OTCase deficiency. Its focus is to increase understanding of structure/function relationships of the OTCase enzyme and the molecular basis of' ornithine transcarbamylase (OTCase) deficiency, an X-linked disorder and the most common inherited defect of ureagenesis. This study will help to define critical domains of OTCase crucial to its normal function. Knowledge of the genetic basis of OTCase deficiency will directly benefit patients and their families in terms of mutation analysis, verification of the deleterious effects of mutations, carrier testing and prenatal diagnosis. This study will help in understanding issues in the inheritance of OTCase deficiency such as the frequency of new germinal mutations, differences in mutation rates between female and male germinal cells and differences in the type of mutations in female vs. male germinal cells. The study has the following aims: (1)To continue to characterize defects in the ornithine transcarbamylase genes in a large patient population with OTCase deficiency. All ten exons of the OTCase gene from these patients will be amplified by the polymerase chain reaction (PCR) and each of the exons will be screened for mutations by single-strand conformational polymorphism (SSCP). The mutations will be defined by DNA sequencing of the "SSCP abnormal" exons. Subsequently, using PCR engineered mutagenesis, expression studies of interesting mutations will be performed. (2) To perform structure/function studies of recombinant "wild type" human OTCase, purified to homogeneity, in order to define its three dimensional structure and functional domains. (3) To express two mutant OTCase genes (Rl4IQ and R277W) in B. coli, purify the mutant proteins and compare their function and structure with those of the wild type enzyme in order to understand how these mutations alter function. The ultimate goal is to obtain crystals of all three proteins and determine their crystal structures. The long term goal of this project is to better understand the functional structure of OTCase and to help families with OTCase deficiency with accurate diagnosis, reliable carrier testing and prenatal diagnosis through a better understanding of the effects of the mutations on OTCase structure and function.